The present invention relates to identifying and isolating genes that encode restriction endonucleases and genes that encode methyltransferases.
Restriction endonucleases originally were identified by studying the inability of bacteriophage from strains of E. coli to infect other strains of E. coli. (Dussoix and Arber, J. Mol. Biol. 5: 37 (1962)). It now is well established that this phenomena, called restriction, is mediated by two types of enzymes, which are called restriction endonucleases and DNA methyltransferases.
Restriction endonucleases primarily cleave double-stranded DNA. Methyltransferases transfer a methyl group from a methyl donor co-factor such as S-adenosyl-L-methionine to a purine or pyrimidine primarily in double-stranded DNA. Methylation by the methyltransferase prevents cleavage of the DNA by the cognate restriction endonuclease. Endogenous DNA of the organism thus is protected by the methyltransferase from damage by the restriction endonuclease. Exogenous DNA entering the cell, which has not been exposed to the methyltransferase, is generally not properly modified and is degraded by the restriction endonuclease.
The two types of enzymes working together function as a simple immune system. The methyltransferase "marks" endogenous DNA as "self" by a sequence-specific pattern of methylation that renders the DNA "immune" to cleavage by the endonuclease. DNA that is not properly modified is seen as "non-self," at least in the sense that it is degraded by the restriction enzyme. Thus, the two cognate enzymes provide a simple mechanism of protecting against the effects of an invading DNA.
In general, methyltransferases and restriction endonucleases form cognate pairs that recognize the same short sequence of DNA with exquisite specificity and require this recognition sequence for activity. Self:non-self recognition in restriction derives from this property of the enzymes. Endogenous methyltransferases modify their genomic DNA at the recognition sites of the cognate restriction endonuclease, protecting it from the endonuclease. The enzymes in different organisms recognize different DNA sequences. Therefore, the methyltransferase from one organism will not protect DNA against the restriction endonuclease from another organism. Sequence specificity allows a single enzymatic mechanism to insulate interacting organisms against the deleterious effects of exogenous DNAs that may be carried between them by vectors or incorporated from the environment. The same sequence specificity is crucial to the practical importance of the enzymes in biotechnology.
The sequences recognized by restriction endonucleases and methyltransferases most often are four or six bases long and centrally symmetric. There are many variations on this basic theme, however, and many five and eight base pair recognition sequences are known, as well as asymmetric recognition sequences and recognition sequences that are located remotely from the modification or cleavage site.
Restriction endonucleases provide the only practical means to cut DNA in a reproducible fashion. Thus, the enzymes have become essential tools in the procedures for isolating, characterizing, studying and expressing genes. In addition, the enzymes now are widely used in clinical and forensic applications in analytic techniques that depend on restriction fragment length polymorphisms. It is likely that the use of these enzymes will increase in the future in both the research setting and commercially as molecular biology research continues its acceleration into marketplace applications. Laboratory and commercial endeavors both will demand variety in the properties of commercially available restriction endonucleases and methyltransferases, which will increase flexibility in using these reagents.
Fortunately, the prokaryotes have evolved a remarkable variety in these enzymes, particularly in their recognition sequences. Two recent compilations list about 1500 restriction endonucleases that have been identified. See Roberts, R, Nuc. Acids Res. 18 Supp: 2331 (1990) and Kessler et al., Gene 92: 1 (1990), both of which are herein incorporated by reference in their entirety. However, only about 200 of the 1500 cataloged restriction endonucleases are readily available through commercial channels.
The limited availability of the enzymes is attributable largely to difficulties producing the enzymes from many organisms and to the shortcomings of alternative production techniques that depend on gene cloning and expression.
Difficulties associated with purifying enzymes from the natural host range from safety problems to low yields. For instance, a variety of organisms that produce potentially useful restriction endonucleases and methyltransferases are human pathogens. Culturing these organisms to purify the enzymes poses a risk to human health that interferes with commercial manufacture.
Even when safety is not a concern, the natural sources of many restriction enzymes cannot provide economical yields. Some potential sources of the enzymes grow only under exotic conditions. Others simply grow very poorly. Still others produce vanishingly small amounts of the enzymes. Even when the growth characteristics of an organism are favorable, it often is difficult to purify the restriction enzymes, due to protease activities or contaminants that are hard to remove, for instance. Another complication of this type occurs in trying to purify the enzymes from organisms that produce several restriction endonucleases. Haemophilus aegyptius produces three restriction endonucleases, for instance.
Several techniques have been developed to overcome the limitations imposed on enzyme production by disadvantageous properties of a natural host. Universally, these methods involve cloning genes that encode restriction endonucleases and methyltransferases and then expressing the genes in a host having properties suitable to enzyme production and purification. Wilson (1988) and Lunnen (1988) have reviewed these methods in some detail. (Wilson et al., Gene, 74: 281 (1988) and Lunnen et al., Gene 74: 25 (1988), both herein incorporated by reference in their entirety). Nonetheless, as discussed below, no universally practical method has yet been developed to clone these genes.
Conventional cloning techniques have been used to clone genes that encode restriction endonucleases and methyltransferases, with some success. As a general rule, these techniques require an effective probe to fish the desired gene out of a genomic or cDNA library. Relatively large amounts of enzyme and considerable investment, effort, expertise and time are required to develop effective immunological or hybridization probes for these genes. Conventional cloning strategies therefore have been used to clone only a few restriction enzymes of manifest economic or scientific interest.
A number of procedures designed specifically to clone genes that encode restriction enzymes have been developed to overcome the problems associated with conventional cloning techniques that require large amounts of protein and immunological reagents or sequence information.
In one technique of this type, DNA (or, potentially, cDNA) from the natural source of the enzyme is shotgun cloned and expressed in recombinant host cells. The cells are screened by exposing them en masse to infection by a bacteriophage, i.e. by restriction. Cells that express a restriction endonuclease should survive the infection and proliferate. This strategy was used to clone the restriction endonucleases HhaII and PstI, as described, respectively, in Mann et al., Gene 3: 97 (1979) and Walder et al., Proc. Nat'l Acad. Sci. USA 78: 1503 (1981).
This strategy suffers from several disadvantages that prevent it from serving as a generally effective method for isolating genes that encode restriction endonucleases and methyltransferases. For one thing, selection by restriction is "leaky," resulting in a high background of false positives. In addition, a resistance to bacteriophage infection is not mediated exclusively by the presence of a cloned restriction endonuclease but can arise from a variety of other factors. A variety of host cell mutations, for instance, can confer resistance, as described in Nwanko et al., Mol. Gen. Genet., 209: 570 (1988). Thus, the method provides only a collection of DNAs which must be characterized further to identify any cloned methyltransferases or restriction endonuclease gene that may be present among them.
An indirect strategy has been developed for cloning restriction endonuclease genes by a methylation protection assay. This technique utilizes in vitro digestion to select DNA encoding a methyltransferase. Since genes that encode a cognate restriction endonuclease and methyltransferase generally are closely linked in the natural host, cloned DNA fragments that express a methyltransferase usually also encode at least a part of the cognate methyltransferase. (Linkage of cognate restriction endonuclease and methyltransferase genes is discussed in Wilson, Nuc. Acids Res. 19: 2539 (1991)).
In carrying out the strategy, DNA from the natural source of the restriction enzyme is shotgun cloned and expressed in a suitable host. The cloned DNA is reisolated from the host cells, pooled and digested in vitro by the restriction endonuclease of interest. The restricted DNA then is transformed into a host and cultured. At best, the only cloned DNAs that will be viable after in vitro restriction endonuclease digestion will be those that expressed the cognate methyltransferase in the transformed host cells. Thus, colonies that arise from the in vitro restriction products should encode the methyltransferase and at least a portion of the linked restriction endonuclease. The procedure originally was described by Borck et al., Mol. Gen. Genet. 146: 199 (1976) and used to clone the gene encoding the hsdRSM.sub.k endonuclease.
The procedure suffers from several drawbacks, however. First, DNAs that do not contain a restriction site recognized by the endonuclease in the in vitro digestion will not be inactivated. Four base pair recognition sequences are likely to be present in almost all DNAs but the false positive background from siteless DNA increases with the length of the endonuclease recognition site.
In addition, not all cognate pairs of restriction endonucleases and methyltransferases are closely linked, and even closely linked genes may be separated during shotgun cloning. Thus, many times clones will encode the methyltransferase but not the restriction endonuclease.
Finally, methyltransferase expression often will not be adequate to protect the heterologous host against the restriction endonuclease. The adverse affect of the restriction enzyme on these cells in many cases will eliminate them from the population before they form discernable colonies. In this event, the method will not be useful for cloning the restriction endonuclease.
Another technique for cloning genes that encode restriction endonucleases is designed to avoid the difficulties that occur when expression of a cloned methyltransferase is not adequate to protect the host against the deleterious effects of the cognate restriction endonuclease. One version of this approach is to shotgun clone DNA from the source organism into a protected host and then screen individual colonies for expression of the endonuclease. The method generally involves transforming a host to express a methyltransferase adequately to protect the host chromosomal DNA against the cloned restriction endonuclease. DNA from the source organism is shotgun cloned into the protected host. Clones that contain the restriction endonuclease gene are identified by screening crude extracts of colonies from the shotgun library for endonuclease activity in vitro in DNA digestion assays. The technique has been used to clone genes that encode the BamHI and DdeI restriction endonucleases, as described by Brooks et al., Gene 74: 13 (1988) and Howard et al., Nuc. Acids Res. 14: 7939 (1986), respectively.
In a modification of this technique, a heterologous methyltransferase gene is used to protect the host cell DNA from nucleolytic attack by the cloned restriction endonuclease. In this approach, a non-cognate methyltransferase gene is expressed in the host before introduction of the shotgun cloned DNA from the source organism. The technique has been used to clone the genes that encode the restriction endonucleases FspI and HaeIII, as described in Wilson and Meda, U.S. Pat. No. 5,179,015.
Both methods suffer from two significant disadvantages, among others. First, a gene encoding a protecting methyltransferase must be available or must be separately obtained to construct a protected host. Furthermore, only individual colonies or small pools of colonies can be screened in this approach for the presence of in vitro restriction endonuclease activity. Thus, the approach cannot be applied where a suitable methyltransferase gene cannot be obtained. Moreover, it is very laborious even when a suitable host expressing the methyltransferase activity is available. It's use therefore has been limited.
Finally, genes that encode restriction endonucleases can be cloned using a temperature sensitive restriction endonuclease that cleaves methylated DNA, as described in Piekarowicz et al., Gene 74: 233 (1988) and Piekarowicz et al., J. Bac. 173: 150 (1991). According to this method, a shotgun library of DNA from the natural source of a restriction enzyme is transformed under permissive conditions into a host that is temperature sensitive for a methylation-dependent endonuclease. After growth under permissive conditions replicates are shifted to non-permissive conditions. DNA in cells that express a cloned methyltransferase will be methylated and will be adversely affected by the activity of the methylation dependent endonuclease.
The method has been demonstrated only in a model system. In this example, a clone encoding the HaeIII methyltransferase was introduced into an E. coli host temperature sensitive for mcrB, a methylation dependent endonuclease of the E. coli mcr restriction system. (The E. coli mcr and mrr digest DNA methylated at specific sequences, as described in Raleigh et al., Proc. Nat'l. Acad. Sci., 83: 9070 (1986) and Dila et al., J. Bac. 172: 4888 (1990)). Transformed host cells were grown initially under permissive conditions at 42.degree. C., where mcrB is inactive, providing for expression of the HaeIII methyltransferase and resultant methylation of the host DNA. Then, replicates were grown at the non-permissive temperature, 30.degree. C., where mcrB is active. Under non-permissive conditions at 30.degree. C., the mcrB endonuclease degraded host DNA methylated by the HaeIII methyltransferase causing the death of cells that expressed this gene. The method suffers from the drawbacks of other methods that select for the methyltransferase.
In sum, there is great interest in restriction endonucleases and methyltransferases. They are employed widely in research and in commercial applications. Cloning the genes that encode these enzymes is key to increasing the variety, quality and economy of commercially available restriction endonucleases and methyltransferases. Producing the enzymes by expressing these genes in a heterologous host offers considerable practical advantages. It avoids the safety problems posed by pathogenic sources of useful enzymes. It circumvents disadvantageous growth habits of the natural host. It vitiates endogenous factors that cause low yields from the natural source. Furthermore, standard expression systems offer significant advantages in reliability, batch to batch uniformity, and economy. Production by cloned genes promises to expand the variety and improve the specific activity, concentration, purity and overall quality of restriction endonucleases and methyltransferases available in the marketplace. Thus, there is a continuing need for improved methods to identify and isolate genes that encode these enzymes.